The objective of the proposed research is to resolve the biological roles and biochemical activities of two transcription factors expressed in the mammalian brain. Both proteins are members of the bHLH-PAS family, respectively designated neuronal PAS domain protein 1 (NPAS1) and NPAS2. NPAS1 is expressed exclusively in neurons of the brain and spinal cord. NPAS2 is expressed predominately in the brain and spinal cord but also in epithelial cell associated with the gut, uterus, mammary gland and prostate. A three-pronged approach will be employed to study the biology and biochemistry of these transcription factors. The first prong of attack will involve targeted disruption of the genes encoding NPAS1 and NPAS2 in laboratory mice. Conventional methods have already been used to uniformly eliminate NPAS1 function. Such mice are viable and an appropriately sized colony is being bred for behavioral and neuro-anatomical studies. NPAS2 function will be conditionally eliminated in each tissue known to express this transcription factor. Mice lacking NPAS2 in specific tissues will be investigated in behavioral, neuro-anatomical and physiological studies of brain tissue in response to both global ischemia and excitotoxic drugs. The second objective of the proposed research will entail efforts to identify NPAS1 and NPAS2 target genes. Cultured neuronal cells will be programmed to conditionally express either NPAS1 or NPAS2 in response to defined stimuli. Messenger RNA will be prepared from such cells before and after induction of each transcription factor. This material will then be subjected to representational difference analysis (RDA) in order to search for genes that are either activated or repressed following induction of NPAS1 or NPAS2. The RDA method will also be utilized to analyze messenger RNA prepared from brain tissue derived from mutant mice lacking either NPAS1 or NPAS2. Once putative target genes have been identified, established methods of molecular analysis will be employed to search for NPAS1 and NPAS2 response elements. The third and final experimental approach will entail biochemical and biophysical studies focused on the mechanisms regulating activation of NPAS2. NPAS2 exists in a latent, cytoplasmic state in pyramidal neurons of the hippocampus and cerebral cortex. As such, there is reason to anticipate that the activity of the protein as a functional transcription factor will require a specific inductive event. Biochemical studies of recombinant NPAS2 have revealed the presence of a heme prosthetic group associated with the PAS domain. Biochemical, molecular biological and biophysical studies will be employed to study the functional relevance of the heme prosthetic group as well as its role in controlling the activity of NPAS2 in pyramidal neurons.